Differential Effects of Synthetic Media on Long-Term Growth, Starch

www.nature.com/scientificreports OPEN Diferential efects of synthetic media on long-term growth, starch accumulation and transcription of ADP-glucosepyrophosphorylase subunit genes in Landoltia punctata Chokchai Kittiwongwattana Murashige & Skoog (MS) and Hoagland’s media were previously used for in vitro culture of Landoltia punctata. During subsequent ex vitro culture, the use of MS medium resulted in a higher growth rate, compared to Hoagland’s medium. Thus, a higher starch content of L. punctata in MS medium was previously hypothesized. Here, L. punctata strain 5632 was isolated and characterized using morphological characteristics and the atpF-atpH intergenic region. During early cultivation stage, fresh weight and relative growth rate in MS medium were lower than Hoagland’s medium. Conversely, starch content in MS medium was considerably higher than in Hoagland’s medium. Medium efects on expression of genes coding for starch-biosynthesis ADP-glucosepyrophosphorylase (AGPase) were determined. Genomic fragments of small (LeAPS) and large (LeAPL1) AGPase subunits were characterized. Diferential expression between each AGPase subunit genes was observed in both media. Additionally, in MS medium, the highest correlation coefcients between starch content and gene expression was found with LeAPS (0.81) and followed by LeAPL3 (0.67), LeAPL2 (0.65) and LeAPL1 (0.28). In Hoagland’s medium, the coefcients of LeAPL3 (0.83) and LeAPL2 (0.62) were higher than LeAPS (0.18) and LeAPL1 (−0.62). This suggested diferent levels of contributions of these genes in starch biosynthesis in both media. Starch functions as an important energy reserve in plants1. During photosynthesis, carbon compounds are generated and converted into glucose that serves as the precursor for starch formation1. Tere are three com- mitted steps in the process1. Te frst one is the generation of ADP-glucose. Secondly, the glucosyl moiety of ADP-glucose is linked to an existing glucan chain through the formation of the α(1-4) linkage, resulting in an extension of the starch chain. Finally, branching of the chain is formed through the formation of the α(1-6) linkage between glucosyl moieties and the chain. ADP-glucose pyrophophorylase (AGPase) is responsible for the formation of ADP-glucose that was proposed as the rate-limiting step2. In higher plants, AGPase is heterotetrameric and consists of two small and two large subunits3,4. Small subunits play the catalytic role, while large subunits mainly function in regulating the enzyme activity5,6. Genes that code for large and small subunits were previously identifed in various dicotyledonous and monocotyledonous plants, e.g., Arabidopsis7, potato8,9, chickpea10, maize11,12 and wheat13,14. Additionally, overexpression of AGPase large subunit genes increased starch content in maize and wheat grains12,15. Another study showed that a transgenic wheat line, overexpressing an AGPase small subunit gene, accumulated starch at a level substantially higher than the wild-type cultivar6. Duckweeds are small aquatic plants that belong to the family Lemnaceae16. Tus far, 37 duckweed species were afliated with the family and classifed into fve genera, including Spirodela, Landoltia, Lemna, Wolfella and Wolfa 16,17. Fronds are leaf-like and function as both vegetative and reproductive organs18. L. punctata has gained the interest in research communities, because of its high starch content that could be used in bioethanol production19–21. Several advantages of L. punctata over other energy crops were also cited22. Tese included rapid growth rate, high starch content and low levels of fber and lignin content22. Additionally, the use of L. punctata Department of Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand. email: [email protected] SCIENTIFIC REPORTS | (2019) 9:15310 | https://doi.org/10.1038/s41598-019-51677-w 1 www.nature.com/scientificreports/ www.nature.com/scientificreports for the energy industry did not compete for land with food crops23. Various external and internal factors afected starch biosynthesis of L. punctata. Nutrient starvation induced starch accumulation4, while phytohormones were internal regulators of starch biosynthesis24,25. Expression of L. punctata AGPase subunit genes, including LeAPS, LeAPL1, LeAPL2 and LeAPL3, were also afected by growth conditions4,25. Expression of LeAPL2 under nitrogen starvation was relatively lower than that under phosphorus starvation4. In contrast, nitrogen defciency induced an early response of LeAPL3, while phosphorus deprivation resulted in an upregulation of LeAPL3 at a later phase4. A transcriptomic study showed that, when L. punctata was treated with uniconazole, expression of two AGPase large subunit genes and starch content were concurrently increased25. Synthetic media were generally used for culturing duckweeds under controlled environments26,27. Medium compositions had various physiological impacts on duckweeds. In Spirodela polyrhiza, exogenous addition of abscisic acid (ABA) in culture medium induced a morphological transition of fronds to turions, a starch accu- mulating structure28. SpAPL2 and SpAPL3, encoding two diferent AGPase large subunits, were also upreg- ulated under such conditions, whereas SpAPL1 expression was not signifcantly increased28. For L. punctata, biomass production was higher when plants were cultivated in Hoagland’s medium for 13 days, compared to MS medium26. However, afer transferred to nutrient-limited pond water for ex vitro culture, L. punctata obtained from MS medium grew 17.1% faster than those from Hoagland’s medium. It was previously hypothesized that cultivation in MS medium resulted in higher starch accumulation that subsequently promoted ex vitro growth26. Tis hypothesis was tested here. L. punctata strain 5632 was newly isolated and characterized, based on its mor- phologies and the atpF-atpH intergenic region sequence. Strain 5632 was long-term (35 days) cultured in MS and Hoagland’s media to observe its biomass production and starch accumulation. Te cDNA fragments of LeAPS and LeAPL1, coding for AGPase large and small subunits, respectively, were cloned and compared with the refer- ence sequences of L. punctata strain 02024. Genomic fragments of both genes were also cloned, and their introns and exons were characterized. Expression levels of four AGPase subunit genes, including LeAPS, LeAPL1, LeAPL2 and LeAPL3, in both media were analysed. Correlation coefcients, between AGPase gene expression levels and starch content, were calculated to determine the contribution of each gene in starch biosynthesis, during the cultivation in MS and Hoagland’s media. Results and Discussion Morphological and molecular characterization. Duckweed samples were collected, and an axenic culture on Hoagland’s medium was established from a single mother frond to ensure the genetic similarity among samples throughout the study. Te strain was designated as strain 5632. It was important to note here that the nutrient compositions of Hoagland’s medium were somewhat diferent from the original publication29. Originally, two diferent macronutrient solutions were described for the preparation of Hoagland’s medium29. Te main − diference between them was the nitrogen source. While one solution contained only NO3 , the other one was − + 29 supplemented with both NO3 and NH4 . Hoagland’s solution, used in this study, had nutrient compositions − (Table 1) that were more similar to the latter one. However, there was a distinction in the NO3 concentration in the original solution (1 mM)29 and this study (6 mM). Additionally, iron was supplied as iron tartate in the original publication29, whereas it was given here as chelated iron. Basic morphological characteristics of strain 5632 were examined (Fig. 1). Fronds were infated and oval-shaped. Te upper surface was dark green. Te lower surface was reddish, suggesting the accumulation of anthocyanins in the tissues. Several roots were present on the lower side of the fronds, indicating that strain 5632 was likely a member of the genus Landoltia. Te presence and the number of roots were distinguishing morphological characteristics among the fve genera of the family Lemnaceae30. For example, a single root was present on each frond of Lemna spp., whereas plants in genera Wolfa and Wolfella did not produce roots. In contrast, several roots were present on each frond of members in genera Spirodela and Landoltia31. Te number of roots of the genus Spirodela ranged from 7 to 21, while that of the genus Landoltia ranged from 2 to 732. Additional characteristics, e.g., a medial series of papillae on the upper surface, frond prophyllum, frond nerves and external anther locules, could be used to diferentiate Landoltia from Spirodela32. However, because of their small and highly reduced structures, identifcation of duckweeds at the species levels could be challenging without a special expertise in the Lemnaceae family33. Biochemical and DNA sequence data were previously combined with morphological and anatomical markers to study the phylogeny of the family Lemnaceae34. Te phylogenetic tree confrmed the presence of the paraphyletic subfamily Lemnoideae, consisting of Spirodela, Landoltia, and Lemna, and the monophyletic subfamily Wolfoideae, comprising of Wolfa and Wolfella. Additionally, a DNA barcode, based on the atpF-atpH intergenic region, was developed to aid the characterization of lemnaceous plants33. For identifcation of strain 5632, the atpF-atpH intergenic

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